Abstract

Mitochondrial DNA (mtDNA) mutations cause neurological and multisystem disease. Somatic (acquired) mtDNA mutations are also associated with degenerative diseases and with normal human ageing. It is well established that certain nucleoside reverse transcriptase inhibitor (NRTI) antiretroviral drugs cause inhibition of the mtDNA polymerase, pol γ, leading to a reduction in mtDNA content (depletion). Given this effect of NRTI therapy on mtDNA replication, it is plausible that NRTI treatment may also lead to increased mtDNA mutations. Here we review recent evidence for an effect of HIV infection or NRTI therapy on mtDNA mutations, as well as discussing the methodological challenges in addressing this question. Finally, we discuss the possible implications for HIV-infected persons, with particular reference to ageing.

Introduction

Human ageing is a complex, multifaceted process and recent studies have suggested several mechanisms which may contribute to an acceleration of physiological ageing in HIV-infected persons, including decreased leukocyte telomere length [1], increased T lymphocyte senescence [2,3], decreased serum testosterone [4], increased coronary artery calcification [5] and increased neurodegeneration [6]. Intrinsic ageing describes the notion that there is a fundamental ageing process within humans, which is not simply the sum total of increasing chronic diseases acquired during life. Such a process starts with molecular damage, thence leading to cell, tissue and organ dysfunction, as succinctly reviewed by Kirkwood [7,8]. One of the best characterized tenants of intrinsic ageing in healthy persons is the phenomenon of acquired (somatic) mitochondrial DNA (mtDNA) mutation [9–18]. Given a wealth of data linking HIV infection, antiretroviral therapy and mitochondrial dysfunction, it is plausible that an acceleration of intrinsic ageing might therefore occur in antiretroviral-treated HIV-infected patients through a mitochondrial mechanism.

Mitochondrial DNA maintenance and somatic mtDNA mutation

Human mtDNA is a compact circular genome of 16.5 kb, encoding 13 essential polyproteins of the respiratory chain, the principal intracellular source of energy (ATP) production [19]. Unlike the nuclear genome which replicates only at cell division, mtDNA is continuously turned over throughout life, with a half-life estimated to be 1–10 days in vivo [20]. There is a sole mtDNA polymerase, pol γ, encoded by the nuclear gene, POLG [21,22]. Mitochondria are the principal source of reactive oxygen species (ROS), a natural by-product of oxidative phosphorylation (OXPHOS) reactions. Furthermore, mtDNA is vulnerable to oxidative damage due to its proximity to the inner mitochondrial membrane (the site of OXPHOS), a lack of protective histones and more limited DNA repair mechanisms compared with nuclear DNA (nDNA). This has led to the ‘vicious cycle hypothesis’ whereby mtDNA mutation causes a functional change in a respiratory chain subunit, leading to partial uncoupling of the respiratory chain, the result of which is increased ROS production and further mtDNA mutation [9,23–25]. However, much recent work has also focused on the role of mtDNA replication error in the generation of mtDNA mutations.

There are two main types of mtDNA mutations: large-scale deletion mutations and point mutations. Large-scale deletion mutations are the predominant mutation type within post-mitotic (non-dividing) tissues, such as muscle and neurons [10,26]. As the mitochondrial genome is composed almost entirely of coding sequence, large-scale deletion mutations typically delete several mitochondrial genes and thus have a high likelihood of a functional consequence within the cell. In contrast, large-scale deletion mutations are not readily observed in replicative tissues (such as peripheral blood mononuclear cells [PBMCs] or in tissue culture systems), a phenomenon which is assumed to arise because the resultant cellular OXPHOS defect would lead to a replicative disadvantage for the cell, and thus cells with such mutations would quickly be lost. The means by which deletion mutations appear in the first place remains to be fully explained, but short homologous repeat sequences in mtDNA are likely to play a role [27]. Virtually all such deletion mutations are located within the ‘major arc’ of the mitochondrial genome, between the origins of replication [26]. The prototype example of this is the 13 bp repeat sequence found at each end of the 4977 bp mtDNA ‘common deletion’ mutation (deletes nucleotide positions m.8483–13446). The ‘common deletion’ is so-called because it is the single most commonly observed large-scale deletion mutation.

mtDNA mutations, heteroplasmy and clonal expansion

Cells contain hundreds to tens of thousands of copies of the mitochondrial genome. Therefore, if a somatic mtDNA mutation arises de novo within a cell it will initially be far outnumbered by wild-type mtDNA. The proportion of mutant mtDNA within a cell or tissue is termed the heteroplasmy level. Typically mutant mtDNA has to exceed a heteroplasmy level of approximately 65% to cause a functional defect within a cell [28]. Such cells have overwhelmingly been described as containing a single (‘clonal’) mutant mtDNA molecule at a high heteroplasmy level [26]. However it is theoretically possible that cells might also experience a functional mitochondrial defect due to the accumulation of multiple mtDNA mutations within a single cell, each of which is present at a relatively low heteroplasmy level. How do somatic mtDNA mutations, therefore, reach high levels within cells? Broadly there are three competing theories. Firstly, that mutant mtDNA has a competitive advantage over wild-type mtDNA and therefore preferentially replicates within a cell. Or secondly, that there is no selective advantage to mutant mtDNA, but it can expand simply through the continued turnover of mtDNA throughout the human lifespan. The latter process arises from the consideration that mtDNA is constantly turned over, even in non-dividing cells and has been termed ‘relaxed replication’. The process has been modelled in silico based on empirically derived parameters, such as the in vivo half-life of mtDNA [29,30]. The third and perhaps least likely explanation, is that recurrent new mutation events happen at the same position in the mtDNA genome. These contrasting models have potential implications for the relative timing of mutation formation with respect to the functional cellular defect that ultimately results [31].

The challenges of measuring mtDNA mutation

In various studies of acquired mtDNA mutation, including ageing and neurodegeneration, a variety of measures of mtDNA mutation have been employed, some qualitative (to detect the presence or absence of mtDNA mutations of interest) and some quantitative. There is no accepted ‘gold standard’: all current methods have technical challenges and the approach depends in part on the tissue under study (Table 1). Some methods are technically demanding and may give conflicting absolute measures of mtDNA mutation burden; however all have proven useful in the comparative analysis of patient/experimental groups [32]. In keeping with developments in HIV sequencing, recently there has been great interest in the application of massively parallel (‘next generation’) re-sequencing (NGS) to the question of mtDNA mutations [33–35]. Deep sequencing of mtDNA by NGS offers relatively high depths of resolution, combined with the great advantages of massively higher throughput and very broad coverage of the mtDNA genome. Limitations include the need to isolate mtDNA from total cellular DNA and the need for PCR amplification which will inevitably introduce some error, as well as the bioinformatics challenges in data analysis.

mtDNA mutations in ageing and disease

Primary mtDNA disorders arise due to mutations within the mitochondrial genome, as single base substitutions (for example mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes [MELAS], m.3243A>G) or single large-scale deletions. In contrast, the disorders of mtDNA maintenance comprise mutations of nuclear genes which directly or indirectly affect mtDNA replication and maintenance. The prototype example is defects in POLG, encoding pol γ, and dozens of mutations have now been described within this gene [21]. These disorders are both genotypically and phenotypically diverse, but in all cases the disease is felt to be caused by the secondary effects on mtDNA and a consequent disruption of cellular OXPHOS function. In many cases, disease is of late onset (in middle age). In these cases the secondary mtDNA defect takes the form of somatic mutations in mtDNA. These disorders predominantly affect neuromuscular tissues, which have a high energy (ATP) requirement, and are composed mainly of post-mitotic cells. In these tissues the predominant mtDNA mutations are large-scale deletions and in diagnostic practice such disorders are often screened for by detecting the presence of multiple large-scale deletion mutations within mtDNA extracted from skeletal muscle biopsy.

mtDNA has a mutation rate estimated to be approximately 5–15× that of the nuclear genome and somatic mtDNA mutations are well described in normal human ageing [9,10,14,25,26,36,37]. Ageing individuals have been shown to have a gradual accumulation of cells which show a functional defect of mitochondrial cytochrome c oxidase (COX) in multiple tissues (including skeletal muscle, cardiac muscle, neurons and colonic crypts) [13,38–41]. Such cells contain high proportional levels of somatic mtDNA mutations which are unique (clonal) within each cell. The degree to which such mtDNA damage is causal in the human ageing process remains a subject of much research, however recent supporting evidence has come from elegant mouse models, where a proof-reading deficient pol γ leads to accelerated accumulation of mtDNA mutations and a prematurely aged phenotype [42,43]. Finally, somatic mtDNA mutations have been linked with a range of common degenerative diseases of ageing. For example brain tissue shows accumulation of somatic mtDNA mutations with normal ageing, but these are further increased in Alzheimer’s disease [17,44–47].

Antiretroviral therapy and the polymerase γ hypothesis

Early in the antiretroviral era it was recognized that a significant minority of patients experienced serious acute or sub-acute treatment toxicity such as symptomatic lactic acidosis and hepatic steatosis, which resembled severe inherited mitochondrial disease [48]. Such acute effects were generally reversible with cessation of the causative drug [49]. Later it was proposed that more common and insidious treatment complications such as peripheral neuropathy and lipodystrophy may also be mitochondrially mediated [50,51]. Mechanistically, it became apparent from in vitro data that many of the older NRTIs cause inhibition of pol γ, manifesting in vitro and in vivo as a reduction in cellular mtDNA content (depletion) [51–63]. This biochemical effect arises in the same manner as the therapeutic effect of NRTIs on HIV reverse transcriptase (HIV RT), namely chain termination during DNA synthesis. The affinity of NRTIs for mtDNA pol γ has been estimated to be approximately 500-fold less than that for HIV RT; however this varies between specific drugs. The hierarchy of pol γ in vitro inhibition is described as: ddC>ddI>d4T>AZT=3TC>ABC=TDF (zalcitabine, didanosine, stavudine, zidovudine, lamivudine, abacavir, tenofovir), a notion which fits reasonably well with in vivo data on observed severity of mtDNA depletion and with the frequency of observed treatment complications [52]. Of the NRTIs in current common clinical usage, both ABC and TDF (TDF being a nucleotide RTI) are reported to have minimal effect on mtDNA replication [64–67].

mtDNA mutations and antiretroviral therapy

It follows from the polymerase γ hypothesis that if the causative NRTI is removed, then mtDNA levels will recover, and indeed there is in vivo data to support this [66]. Although most patients in industrialized countries are now treated with ABC- or TDF-based combination antiretroviral therapy, there are large numbers of patients who have had many years of exposure to the pol γ inhibiting NRTIs (for example, ddC, ddI, d4T and AZT) in the past. These patients will not show persistent mtDNA depletion [35]. If there is any persistent effect on mtDNA in such patients it is likely to be a qualitative defect: somatic mutations. Based upon our knowledge of the action of NRTIs, coupled with our understanding of mtDNA mutation formation, there are several hypothetical means by which NRTI therapy could promote mtDNA mutations (as discussed in What is driving NRTI-induced mtDNA mutations?). Studies examining mtDNA mutations in the setting of antiretroviral therapy are presented in Table 2. These studies have used a wide variety of methods and have studied a variety of tissues, which may be expected to support different mutation types. Analysis of these data reveals some contrasting findings. The first published example of mtDNA mutations in an NRTI-treated patient was a fatal case of lactic acidosis where large-scale mtDNA deletions were seen in the liver [68]. It is unclear whether the drug exposure may have ‘unmasked’ an underlying inherited mitochondrial DNA maintenance disorder [68]. The first systematic evidence that NRTIs may indeed lead to increased somatic mtDNA mutation came from a longitudinal study of patients commencing d4T therapy [69]. Surprisingly, 5 of 16 subjects developed new mtDNA mutations in PBMCs after starting therapy [69]. These observations have been revisited recently when a small series of papers have sought to detect antiretroviral-associated acquired mtDNA mutations in a variety of settings. Several of these studies have suggested an increase in mtDNA mutations: in humans [35,69–74], rodents [75–78] and in vitro [79]. One recent study (a longitudinal analysis of blood samples from thymidine analogue treated patients) has failed to replicate these findings, although it is possible that low-level mutations might have been missed [80]. A few studies have specifically looked for large-scale mtDNA deletion mutations within post-mitotic tissues [35,72]. As described earlier, these are expected to be the most functionally significant mutations, due both to the genetic nature of the defect and the tissues affected. For example, in our own study, we have demonstrated that patients with a history of exposure to pol γ inhibiting NRTIs show an excess of skeletal muscle fibres containing high levels of somatic mtDNA mutations (both point mutations and large-scale deletion mutations) [35]. Importantly the relevant drug exposures were often in the remote past, suggesting that the mutations are indeed irreversible.

What is driving NRTI-induced mtDNA mutations?

The intuitive deduction when considering mechanisms whereby antiretroviral therapy may increase somatic mtDNA mutations is accelerated de novo mutagenesis. This is essentially the correlate of an inherited POLG defect, whereby there will be continuous production of higher levels of mtDNA mutations throughout a treatment period. There are plausible biological mechanisms whereby this might occur, for example impairment of the limited exonuclease function of pol γ by NRTIs would lead to poor proofreading. This biochemical effect has been reported at least for AZT [52]. Furthermore it is suggested that, in addition to any effects on pol γ, AZT may inhibit mitochondrial thymidine kinase 2 (TK2) plausibly leading to purine/pyrimidine imbalance and thus mtDNA mutations [81,82]. The difficulty with this de novo mutagenesis hypothesis however, is that such mutations will need to undergo clonal expansion before they achieve high enough levels within cells to cause a functional defect. Under a simple ‘relaxed replication’ model (as described above), these mutations will be predicted to take many years or decades to reach high levels within cells. It would therefore be predicted that a typical period of NRTI exposure during early adult life would lead to a functional cellular defect only in late middle age, if ever (Figure 1). Furthermore, modelling data suggest that the relative increase in mutation rate would need to be very substantial indeed to cause a significant increase in cellular mitochondrial (COX) defects [30].

Figure 1. Early mutation hypothesis, clonal expansion and putative role of NRTIs

Diagrams show an individual cytochrome c oxidase (COX)-positive cell (brown) in a young individual eventually becoming COX-deficient (blue) in later life, over a representative timescale. Multiple copies of wild-type mitochondrial DNA (mtDNA) are initially present within the cell, shown as green circular molecules (in reality hundreds to thousands per cell). A mutation occurs (red molecule) which is initially at a low percentage heteroplasmy level within the cell, but over time may clonally expand to reach a high heteroplasmy level causing the COX defect. (A) Under an ‘early mutation’ hypothesis, new mtDNA somatic mutations that lead to COX-deficient cells late in life, arise in early life and clonally expand very slowly, by a non-selective process of drift (‘relaxed replication’). (B) Under an alternative model, mutant mtDNA clonally expands relatively rapidly within cells, and therefore the observed COX defect in late life is due to a relatively recent mutation. Such a process of rapid clonal expansion is likely to require a selective replicative advantage for mutant mtDNA. A hypothesis of accelerated clonal expansion of mtDNA mutations due to nucleoside reverse transcriptase inhibitor (NRTI) exposure is most coherent with an ‘early mutation’ hypothesis (A) where the somatic mutations have already occurred by early adult life, and therefore expand to result in COX-deficient cells many years earlier than expected.

Therefore, we have proposed an alternative hypothesis of accelerated clonal expansion [35]. This hypothesis requires no additional de novo mutagenesis, as clonal expansion can act on the pre-existing somatic mtDNA mutations which naturally arose early in life. Modelling this scenario suggests that a finite period of mtDNA depletion associated with NRTI therapy, leads to accelerated molecular segregation and thus accelerated clonal expansion of pre-existing (age-associated) mtDNA mutations. The severity of mtDNA depletion will predict the rapidity of clonal expansion, and this is in keeping with our empirical data whereby potent pol γ inhibitors (ddC, ddI) which cause profound mtDNA depletion, caused a much higher proportion of COX-deficient fibres (that is, more rapid clonal expansion) than weaker inhibitors (d4T, AZT). Importantly this model acts rapidly during a period of NRTI treatment (as the ‘seeding’ mutations are already present). In conclusion, the resultant COX defect is predicted to appear rapidly during the causative NRTI therapy and be persistent thereafter. Finally, a variation on a non-selective clonal expansion model is the idea that NRTI therapy may select for mutant mtDNA. This is certainly plausible in the case of large-scale deletion mutations, where we may suggest that deleted (smaller) mtDNA molecules would replicate more readily in the face of NRTI-induced pol γ inhibition than full-size molecules. There is analogous data to support this notion from the non-HIV setting [83] and we are currently investigating this phenomenon in the context of NRTI therapy. Such a process would ultimately serve to further accelerate clonal expansion.

The functional consequences of mtDNA damage

The evidence that somatic mutations in mtDNA and associated functional cellular (COX) defects are temporally related to the normal human ageing process is increasingly robust, however the key question remains: to what extent they are causally related? The POLG ‘mutator’ mouse would seem to provide evidence for a causal relationship in that, as far as we know, the only difference between this mouse and the wild-type is that it accumulates mtDNA mutations at a significantly increased rate, leading to a progeroid phenotype [42,43]. However, in the case of human POLG defects, or other inherited disorders of mtDNA maintenance, there is also more rapid accumulation of secondary mtDNA defects, but the phenotype is usually one of late-onset neurodegenerative disease, but not premature ageing. One relevant consideration is potentially the very great difference in normal lifespan in humans (>80 years) and mice (<3 years). Although it is not certain whether the rate of mtDNA turnover (mtDNA ‘half-life’) differs significantly between species, it is very likely that the elderly human has experienced far more cycles of mtDNA replication than the mouse. As described earlier, mtDNA turnover is thought to be the ‘engine’ driving clonal expansion of mtDNA mutations. Thus in a long-lived mammal such as the human, a low mutation rate may well still be entirely compatible with a functional role in ageing given the long period of time for those mutations to clonally expand and lead to defects of oxidative function within cells.

Moving away from animal models, is there evidence that humans with healthy mitochondria are less ‘biologically aged’ than humans of equivalent chronological age who have greater somatic mitochondrial defects? Early work from Doug Turnbull’s group suggested that elderly persons with increased physiological performance (for example grip strength), tended to have lower proportional COX defects on skeletal muscle biopsy [39]. Of course, this observation could either be compatible with the notion that slower accumulation of mtDNA defects results in preserved function or that preserved muscular function (for example through better general health and higher levels of exertion), results in the preservation of mitochondrial function. It has also been suggested that exercise may lead to lower mutation levels through gene shifting of dormant myoblasts causing them to re-enter the cell cycle [84]. Further longitudinal studies are desperately needed in this area to better address such questions. However, such studies are difficult to do owing to the vast timescales involved, and the need for repeated biopsies; as described earlier, a COX defect in an individual in their seventh decade might have resulted from a new mutation event several decades earlier. And, as suggested above, shorter-lived mammals are probably not good enough models of this process.

Is there other indirect evidence to support a causal role for mtDNA mutations in ageing? If certain mtDNA genotypes were more or less susceptible to somatic mtDNA mutations, then we might expect to see differences in the rate of ageing between such populations. Work by David Samuels suggests that this notion may hold true when comparing animal species. As described earlier, short mtDNA sequence repeats are strongly associated with the formation of large-scale mtDNA deletion mutations. Samuels demonstrated that species with lower numbers of such homologous repeats in their mtDNA genome show increased longevity [85]. Within the human species, mtDNA is also highly polymorphic and one mtDNA haplogroup (D4a) contains single nucleotide polymorphisms that disrupt that 13 bp repeat associated with the m.δ4977 ‘common deletion’. The D4a haplogroup is unexpectedly over-represented among centenarians [86]. Finally, another polymorphism in the non-coding mtDNA control region, results in a change in the origin of mtDNA replication, with predicted consequences for mtDNA turnover. This group also shows increased longevity [87].

As described in the Introduction, there are multiple biological parameters for which HIV-infected patients show deleterious changes which resemble those expected in uninfected elderly persons [1–6]. Where might mtDNA mutations fit into this picture? The putative downstream consequences of mtDNA somatic mutations at the tissue level are shown in Figure 2. It seems likely that ‘mitochondrial ageing’ in NRTI-treated HIV-infected patients might have a role in driving frailty and perhaps sarcopenia in this patient group. Both these gerontological markers have been shown to be prevalent in HIV-infected patients and in the non-HIV setting are predictive of adverse clinical outcomes [88–91]. Simple clinical measures of ‘ageing’ are lacking and extensive long-term follow-up may be required to establish a clear causal link between markers of intrinsic ageing in HIV and clinical outcomes. These findings of increased mtDNA mutations are probably likely to be of most relevance to those patients with extensive past exposure to pol γ inhibiting NRTIs who are entering older age. Finally, millions of patients in the developing world have been exposed to AZT and d4T in recent years as part of antiretroviral roll-out programmes, and these data are likely to reinforce the WHO position that such patients should be switched to TDF-based therapy as soon as is feasible [92].

Figure 2. Hypothesized model of how age-associated somatic mitochondrial DNA mutations may lead to a functional defect at the tissue level

Somatic mutations may arise either through oxidative damage to mitochondrial DNA (mtDNA), for example, due to reactive oxygen species (ROS) or through natural replication errors. mtDNA mutations may cause synthesis of abnormal respiratory chain proteins, leading to partial uncoupling of the mitochondrial respiratory chain. This may lead to a vicious cycle of increased ROS. In order for somatic mtDNA mutations to cause a functional mitochondrial (cytochrome c oxidase [COX]) defect at the cellular level, the mutation must clonally expand to reach a high percentage level within the cell. Frequent COX-deficient cells will decrease function of the tissue, and may also undergo apoptosis. Boxes shown in red may plausibly be adversely affected by HIV or nucleoside reverse transcriptase inhibitor antiretroviral therapy exposure.

Preventing somatic mtDNA mutation: therapeutic avenues

A rational approach to ameliorate the effects of age-associated mtDNA mutations depends to a large extent on how and when those mutations arise, and what their natural history may be in terms of the progression from a new mutation to a functional defect. Many of these fundamental aspects of mtDNA biology remain the subject of on-going research, and as such, discussion of how best to prevent these changes is currently largely a matter of speculation.

If one takes the view that de novo mtDNA mutations arise at significant levels throughout the normal human lifespan, and that these may clonally expand relatively rapidly (perhaps through positive selection), then prevention of new mutation formation may be of therapeutic benefit. In the case of mutations arising from ROS damage, anti-oxidant treatment might be proposed. Anti-oxidant compounds frequently show beneficial effects in vitro but almost none of these benefits have so far been translated in vivo. There are a few animal models which have suggested a beneficial effect. For example, a mouse model has been developed which expresses a mitochondrially targeted antioxidant. These mice show reduced levels of ROS, reduced mtDNA mutations and increased life span [93]. Alternatively it may be that natural replication errors are of primary importance. Thus, one would wish to either increase the fidelity of pol γ or increase mtDNA repair mechanisms. Currently there are no clear means to achieve either of these aims.

Conversely, we may take the view that clonal expansion is the more important process. As discussed above, there is some recent evidence to suggest the relative importance of clonal expansion in normal human ageing. If de novo mutations are therefore very early events, and clonal expansion is a slow process, then targeting clonal expansion would seem attractive. Indeed, clonal expansion is a necessary step, no matter how slowly or rapidly it occurs. Currently, the key putative means to change the rate of clonal expansion is to modify mitochondrial biomass or mtDNA copy number (the two appear to be intrinsically linked). How may we therefore increase cellular mtDNA content? In the case of in vitro studies this effect has principally been achieved by uridine supplementation [94]. In a rodent model, uridine supplementation has prevented the detrimental effects of NRTI treatment on the brain, presumably by preventing profound mtDNA depletion [95]. However, there is no good data to suggest that uridine supplementation is beneficial in humans, including in the HIV-infected patient [96]. However, the best evidence for an approach of increasing mitochondrial biomass probably comes from exercise studies. It has long been established that endurance exercise increases cellular mtDNA content, principally in a peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α)-dependent manner. Frail elderly subjects show reduced cellular mtDNA content compared with active elderly and younger subjects. This reduction in copy number may plausibly accelerate the clonal expansion of mtDNA mutations within cells in these subjects. Exercise studies have been attempted in patients with inherited mtDNA defects [97]. On serial biopsy, subjects with single deletion mtDNA disorders, showed a decrease in the proportion of COX-deficient fibres [98]. The exercise-treated patients also have increased cellular mtDNA content. Finally, the most compelling evidence perhaps comes from a recent elegant paper using the pol γ ‘mutator’ mouse described previously. When subjected to endurance exercise the homozygous mutant mouse appears phenotypically as the wild-type mouse, rather than developing the progeroid state. mtDNA content is significantly increased in the mouse subjected to endurance exercise. However, the mutant mtDNA proportion remains comparable with that observed in the homozygous mutant mouse that did not undergo endurance exercise [99]. This suggests that by increasing mitochondrial biomass, endurance exercise unfortunately cannot shift the proportional balance of wild-type and mutant mtDNA (and in fact it therefore increases the total amount of mutant mtDNA), but it can perhaps prevent the functional consequences of mutant mtDNA on the cell. These theoretical concepts, as applied to HIV infection and antiretroviral therapy are shown in Table 3.

Conclusions

There are numerous ways in which many of the older NRTI antiretroviral drugs may increase somatic mutations in mtDNA and there is increasing empiric evidence to suggest that this phenomenon does indeed occur in treated patients. It is plausible that one of the mechanisms may be via an acceleration of the clonal expansion of pre-existing (age-associated) mtDNA mutations. Fuller understanding of the exact mechanism involved has potential implications for predicting the natural history of such mutations as patients continue to age. Conversely, NRTI exposure arguably presents a rather unique scenario in which aspects of normal mtDNA maintenance are iatrogenically altered. Observations from NRTI treatment may further the debate on the fundamental biology of mtDNA mutations in normal human ageing.

Disclosure statement

PFC is a Wellcome Trust Senior Fellow in Clinical Science (101876/Z/13/Z) and a UK NIHR Senior Investigator. PFC receives additional support from the Wellcome Trust Centre for Mitochondrial Research (096919Z/11/Z), the Medical Research Council (UK) Centre for Translational Muscle Disease research (G0601943), and EU FP7 TIRCON, and the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based at Newcastle-upon-Tyne Hospitals NHS Foundation Trust and Newcastle University. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. BAIP was funded by the Medical Research Council, UK (G0800470). KG is funded in part by AstraZeneca. The authors declare no competing interests.

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